Water BacteriologyMethod: Multiple tube fermentation technique (MPN)

A.I. Presumptive test: Day 1

Culture medium: LTB

1. Label 9 test tubes of LTB1-3(3)---LTB 10mL4-6(3)---LTB 1mL7-9(3)---LTB 0.1mL

2. Shake the bottle w/ the H2O , inoculate all 9 LTB tubes using a sterile pipet.

3. Mix all tubes at least 20x.4. incubate at 37C for 24 hours.5. Observe for the presence of gas in the inverted vials.6. submit all (+) tubes to confirmed test.

II. Confirmed Test: Day 2Culture media: BGLB & EC

1. Shake each (+) tubes in the PT.2. Using a sterile wire loop, transfer 1 loopful of culture to BGLB

tubes. (scall (+) LTB tubes to BGLB)

3. Choose 1(+) LTB (10mL, sc to EC medium4. Incubate BGLB at 37C and EC at 44C for 24 hours5. Observe for the presence of gas.

*if (-) reincubate for another 24 hours6. Record all (+) BGLB tubes and determine the MPN.

Example: MPN Dtermination(Most Probable Number of Coliforms)

-determine in the confirmed test (BGLB) after 48 hours of incubation

BGLB 10mLBGLB 10mL (+) turbid w/ gas=3BGLB 10mL

BGLB 1mLBGLB 1mL (+) turbid with gas=3BGLB 1mL

BGLB 0.1mLBGLB 0.1mL (-) no gas=0

BGLB 0.1mL

MPN=3-3-0=240 col./100mL H2O

III. Completed TestCulture media: EMB, LTB & NA slant

*remember: EMB: differential mildly selectivedifferentiates lactose fermenters from non-lactose

fermenters

1. Choose 1(+) BGLB (10mL)=Streak an EMB plate (4Q streaking)2. Incubate at 37C for 24 hours.

a. After 24 hrs incubation. from EMB plate-look for typical colonies (pick 1 or more)-look for atypical colonies (pick 2 or more and SC to

LTB and NA slant)b. Incubate at 37C for 24 hrs.c. Observe for gas production in LTB tubed. Make a smear---do Gram stain from NA slant